PROJECT SUMMARY/ABSTRACT Serotonin (5-HT) signaling in brain reward circuits modulates drug-seeking behavior. Specifically, 5- HT6 receptors provide excitatory signals to medium spiny neurons that make up the direct and indirect pathways, the two main outputs from the nucleus accumbens (NAc). These receptors are localized to the primary cilia of neurons, signal through type 3 adenylyl cyclase (AC3), and play a key role in drug reward and reinforcement. How the 5-HT6 receptor and its downstream signaling components influence drug taking via reward circuitry remains unclear. Our lab has shown that increased expression of 5-HT6 receptors specifically in indirect but not direct pathway results in a decrease in cocaine self-administration by increasing the sensitivity to the reinforcing properties of cocaine. Thus, 5-HT6 receptors appear to influence cocaine reinforcement in a pathway-selective manner. However, overexpressed receptors may have altered the subcellular distribution and cilia- specific localization of 5-HT6 receptors. The proposed experiments will further test the pathway specificity by knocking out endogenous 5-HT6 receptor signaling in a pathway-selective manner. This proposal seeks to determine the exact nature of how endogenous 5-HT6 receptors regulate cocaine self-administration, and whether cilia-specific signaling plays a role in addiction-like behavior. Aim 1 will create a Cre-dependent CRISPR-Cas9 viral vector targeting the 5-HT6 receptor gene to enable pathway-specific knockout of the receptor with an intersectional viral strategy. Aim 2 will investigate the pathway-selective influence of 5-HT6 receptor signaling on cocaine self-administration in rats by disrupting expression of this receptor in the direct or indirect pathway neurons selectively. Behavioral economic analysis of cocaine taking will deepen our understanding of how these receptors modulate cocaine intake, reinforcement, and motivation. Aim 3 examine the molecular mechanism by which 5- HT6 receptors regulate cocaine-seeking behavior by disrupting AC3 signaling in a pathway-specific manner. This will allow us to validate the link between AC3 and 5-HT6 receptor function, and provide novel insights into the effects of cilia-specific signaling on cocaine self-administration. The novel intersectional knockout approach in this proposal has broad implications for addiction research, as well as neuroscience in general, and provides a training plan that enables the expansion of my current knowledge and repertoire of molecular and behavioral techniques. Together, these three Aims, along with the other elements of my training plan, could inform new treatments for addiction, prepare me to become an independent clinician-neuroscientist, and address my desire to conduct research that spans the translational gap between basic neuroscience and the devastating impact of addiction in families and society.